Regulatory T Cells Function in Established Systemic Inflammation and Reverse Fatal Autoimmunity

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss-of-function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of “redeemed” Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease, and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

protection against them. Thus, Treg cells are functional in settings of established broad spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.
Regulatory T (Treg) cells expressing the X-linked transcription factor Foxp3 have been implicated in the control of inflammation in diverse settings [1][2][3][4][5][6][7][8][9] . Mice and humans lacking functional Foxp3 gene develop fatal multi-organ autoimmune and inflammatory disease featuring lymphadenopathy and splenomegaly, eosinophilia, hyper IgE syndrome, and markedly increased systemic levels of a wide range of pro-inflammatory cytokines 1, 2, 3 . Foxp3 protein expression is required for Treg cell differentiation and function 4,5,6,7 . Analyses of mice harboring a Foxp3 GFPKO reporter-null allele showed that Foxp3-deficient GFP + Treg "wannabes" lack suppressor capacity 8,9 , which can be restored by expression of a Foxp3 transgene 10 . Foxp3-deficient mice display autoimmune pathology by day 10 of life with most mice dying within 3-4 weeks 5,11,12 . Likewise, Treg cell ablation induced upon diphtheria toxin (DT) treatment of healthy adult Foxp3 DTR-GFP mice, whose endogenous Foxp3 locus encodes a simian DT receptor (DTR)-GFP fusion protein, leads to similar widespread autoimmune inflammation to which they succumb within 2 weeks 13 . Adoptive transfer of wild-type Treg cells into Foxp3 DTR-GFP mice at the time of DT administration or into 1-day-old Foxp3 mutant mice prevents the disease 5,13 . These studies have unequivocally demonstrated a critical role of Treg cells in preventing autoimmune inflammation and associated pathologies.
However, a major outstanding issue is whether Treg cells can effectively function in settings of established systemic inflammation and reverse and restrain severe autoimmune diseases. In fact, a large body of work in experimental animal models of infection, inflammation, and cancer suggests that major immune effector cytokines, including IL-6, IL-1, type I and II IFNs, IL-23, and IL-4, can cause Treg cells to lose Foxp3 expression, become functionally inactivated, and acquire pro-inflammatory features, or render immune effector cells refractory to Treg cell-mediated suppression 14,15,16,17,18,19,20 . Accordingly, numerous analyses of Treg cells present at inflammatory sites of autoimmune patients suggested their function was impaired 21,22,23,24,25 . Additional uncertainty stems from the possibility that the three archetypal inflammatory immune responses (types 1, 2, and 3), featuring distinct spectra of secreted and cellular effectors, may differ in their ability to compromise Treg cell functionality or sensitivity to Treg cell-mediated suppression. In this regard, variation in Treg cell frequencies was reported to result in a disproportionate dysregulation of type 2 vs. type 1 autoimmunity 26 . Thus, to address the major outstanding question above, we sought to employ an experimental animal model which would: 1) exhibit systemic mixed type 1, 2, and 3 autoimmunity and tissue inflammation; 2) enable temporal control and efficient switching of Treg cell suppressor function in an established disease; 3) eschew adoptive Treg cell transfer, which does not guarantee their sufficient migration to and accumulation at the inflammatory sites; and 4) allow for extended longitudinal observation.
We reason that the cardinal features of the aforementioned archetypal inflammatory responses are shared by a broad spectrum of autoimmune and inflammatory diseases. The breadth and severity of the autoimmune inflammation resulting from Foxp3 deficiency, characterized by activation and expansion of all major adaptive and innate immune cell types and rampant cytokine storm, make it an ideal experimental setting for testing the ability of Treg cells to operate in established inflammation. By engineering mice where an inducible Cre recombinase allowed for the installation of Treg cells in severely diseased animals, we found that Treg cells were functional in settings of established systemic mixed-type inflammation and capable of restoring health. Upon sensing the inflammatory environment, Treg cells became activated, rapidly expanded, exhibited heightened suppressive capacity, and persisted for an extended period of time with no signs of dysfunction.

Restoration of Foxp3 expression in Treg "wannabes"
To investigate the ability of Treg cells to function in settings of established severe inflammation, we generated mice harboring a reversible Foxp3 loxP-Thy1.1-STOP-loxP-GFP reporter-null allele (Foxp3 LSL ). In these mice, a loxP site-flanked Thy1.1 reporter followed by a STOP cassette was inserted into the Foxp3 locus upstream of the Foxp3 GFP reporter allele with Thy1.1 expression marking a population of Treg "wannabes" (Extended Data Fig.  1a-d). These cells were phenotypically similar to those expressing the Foxp3 GFPKO allele 8 , and distinct from either naïve or activated conventional CD4 T cells, or Foxp3-sufficient Treg cells (Extended Data Fig. 1e). To ascertain that Treg "wannabes" bare a similarly self-reactive TCR repertoire as Treg cells, we assessed their TCR Vβ5 usage. C57BL/6 mice express viral superantigens vSAG8 and vSAG9, whose recognition by Vβ5 utilizing TCRs 27 leads to partial deletion of Vβ5 + conventional CD4 T cells and promotes the generation of Vβ5 + Treg cells 28,29 . We observed that Treg and "wannabe" cells showed comparably elevated Vβ5 usage relative to conventional CD4 T cells. This result suggests Treg and "wannabe" cells were similarly "self-focused" and that lack of Foxp3 did not result in shifted self-reactivity of the TCR repertoire of Treg "wannabes" (Extended Data Fig.  1f). Combining Foxp3 LSL allele with Cd4 creERT2 allele encoding a tamoxifen-activatable Cre-ERT2 fusion protein enabled excision of the STOP cassette upon 4-hydroxytamoxifen (4-OHT) treatment and conversion of Foxp3-deficient Thy1.1 + "wannabes" into Foxp3sufficient GFP + Treg cells ( Fig. 1a and 1b). The latter, when co-transferred with Foxp3 − CD4 T cells into lymphopenic recipients, suppressed the wasting disease similar to those isolated from control Foxp3 GFP mice (Extended Data Fig. 1g). Thus, rescued expression of endogenous Foxp3 in Treg "wannabes" conferred suppressor function and fitness in accordance with previous findings.

Treg cells reverse established disease in young and adult mice
Hemizygous male Foxp3 LSL mice developed multi-organ autoimmune inflammation indistinguishable from that of Foxp3-deficient mice, to which they succumbed within 3-4 weeks with few surviving for up to 6 weeks after birth. To test whether Treg cells can suppress ongoing inflammation, male Foxp3 LSL Cd4 creERT2 or Foxp3 LSL Cd4 wt mice were administered with a single dose of 4-OHT at 2 weeks of age, by which time they exhibited pronounced autoimmune syndrome with conspicuous clinical manifestations including blepharitis and macroscopic skin lesions. The recombination efficiency was comparable in lymphoid and non-lymphoid organs on day 3 following 4-OHT administration (Extended Data Fig. 2a). Within 4 weeks, the disease in 4-OHT-treated Foxp3 LSL Cd4 creERT2 mice was greatly alleviated with the extent of activation of innate and adaptive immune cells approaching the level of healthy Foxp3 DTR-GFP Cd4 creERT2 controls. In contrast, the disease in 4-OHT-treated Foxp3 LSL Cd4 wt mice rapidly progressed with no mice surviving beyond 6 weeks of age ( Fig. 1c-e; Extended Data Fig. 2b-f).
Since the observed reversal of inflammation could be due to restoration of Foxp3 expression in thymocytes or peripheral Treg "wannabes", we combined 4-OHT treatment with continuous FTY720-induced blockade of thymic output. Accordingly, we observed increased numbers of thymic Treg cells and the percentages of Rosa26 loxP-STOP-loxP-tdTomato (R26 Tom ) recombination reporter-expressing thymocytes labeled upon 4-OHT treatment (Extended Data Fig. 3a,b). Diminished immune activation and inflammation in Foxp3 LSL Cd4 creERT2 mice, compared to Foxp3 LSL Cd4 wt controls, on day 14 following combined 4-OHT and FTY720 treatment suggested that Treg cells rescued in the periphery were effective at controlling inflammation in the absence of thymic output (Extended Data  1f). In addition, levels of IgE, IgG1 and IgM were brought down by newly generated Treg cells in Foxp3 LSL Cd4 creERT2 mice in comparison to control Foxp3 LSL Cd4 wt mice ( Fig. 1g, Extended Data Fig. 2g). Manifest tissue immune infiltration and inflammation disappeared in Foxp3 LSL Cd4 creERT2 mice but not Foxp3 LSL Cd4 wt controls within 4 weeks of 4-OHT administration ( Fig. 1 h-i). A closer examination of the skin pathology in pre-treatment Foxp3 LSL Cd4 creERT2 and Foxp3 LSL Cd4 wt mice revealed lichenoid interface dermatitis, which was resolved in treated Foxp3 LSL Cd4 creERT2 mice but continued to worsen in Foxp3 LSL Cd4 wt mice (Extended Data Fig. 4a-b). Additionally, liver damage evident by apoptotic hepatocytes and decreased serum albumin concentrations in Foxp3 LSL Cd4 creERT2 mice normalized within 4 weeks after 4-OHT treatment (Extended Data Fig. 4c-e). The disease reversal was not a mere consequence of subtracting the Treg "wannabes" from the CD4 T cell pool, considering that the disease severity in Foxp3-deficient mice harboring Treg "wannabes" was identical to that of neonates subjected to chronic Treg cell ablation 13 . Thus, Treg cells are capable of effectively suppressing multiple arms of the inflammatory immune response in young mice under conditions of established autoimmunity, causing its reversal.
To assess whether Treg cells can restrain ongoing inflammation in adulthood, we generated healthy mosaic heterozygous female Foxp3 DTR-GFP/LSL Cd4 creERT2 mice, which harbor both a functional Treg and a non-functional "wannabe" population expressing the Foxp3 DTR-GFP and Foxp3 LSL allele, respectively. Recurrent DT administration causes widespread autoimmunity in Foxp3 DTR-GFP mice to which they succumb within 10-14 days 13 . Similarly, DT-treated adult (8-10-week-old) Foxp3 DTR-GFP/LSL heterozygous females exhibited massive immune activation by day 7, at which point the diseased Foxp3 DTR-GFP/LSL Cd4 creERT2 and Foxp3 DTR-GFP/LSL Cd4 wt mice were administered tamoxifen to convert Treg "wannabes" to Treg cells (Fig. 2a). The rehabilitated Treg cells efficiently suppressed T cell activation within 2 weeks and ameliorated the inflammatory lesions in the skin, lung, and liver by week 5, whereas in control Foxp3 DTR-GFP/LSL Cd4 wt mice the fatal disease rapidly progressed ( Fig. 2b-e, Extended Data Fig. 5a-c). The 5 week endpoint was chosen as it was the longest period we were confident the mice were not developing neutralizing DT-specific antibodies. Thus, Treg cells can function under inflammatory settings and suppress established lethal autoimmunity in both neonates and adults.
Notably, activated Treg "wannabes" from DT-treated Foxp3 LSL/DTR-GFP sick female mice shared some gene expression changes with those of activated Treg cells in rescued Foxp3 LSL/y mice (Fig. 3d, Extended Data Fig. 6). However, this acquisition of activated phenotype by Treg "wannabes" did not confer any appreciable suppressive capacity, as Foxp3 LSL/DTR Cd4 wt mice, in which tamoxifen treatment could not restore Foxp3 expression, died of similar systemic multi-organ inflammation seen in Foxp3 LSL/y mice. Thus, the heightened suppressor capacity of rehabilitated Treg cells in Foxp3 LSL Cd4 creERT2 mice could be due to exposure to an inflammatory environment upon Foxp3 induction or a cell-intrinsic effect of delayed Foxp3 expression regardless of the environment. To distinguish between these two possibilities, we analyzed the "wannabe"-converted and control Treg cells under basal non-inflammatory conditions in healthy heterozygous female Foxp3 LSL/WT Cd4 creERT2 and Foxp3 DTR-GFP/WT Cd4 creERT2 mice, respectively, on day 7 post 4-OHT treatment. In contrast to Treg cells exposed to the inflammatory environment in male mice, Thy1.1 − GFP + Treg cells in 4-OHT-treated mosaic Foxp3 LSL/WT Cd4 creERT2 females failed to exhibit increased proliferative activity or enhanced CTLA4 and GITR expression, compared to GFP − Foxp3 + Treg cells expressing the Foxp3 WT allele in the same mice (Fig.   3f). Likewise, comparable suppressor activity was observed for Thy1.1 − GFP + Treg cells and control GFP + counterparts isolated from 4-OHT-treated healthy Foxp3 LSL/WT Cd4 creERT2 and Foxp3 DTR-GFP/WT Cd4 creERT2 female mice, respectively (Fig. 3g). To the contrary, sick Foxp3 DTR-GFP/LSL Cd4 creERT2 mosaic female mice, subjected to DT-induced ablation of Foxp3 DTR-GFP Treg cells and tamoxifen-induced Foxp3 restoration in Foxp3 LSL cells, showed a transient increase in Treg percentages at 1 and 2 weeks post tamoxifen-mediated rescue, which disappeared by week 5 (Fig. 3h). These Treg cells also exhibited enhanced proliferation and heightened activation as evidenced by elevated expression of Ki67, CTLA4, GITR and ICOS (Fig. 3i, j). Thus, the enhanced proliferative potential and suppressive capacity of Treg cells in diseased Foxp3 LSL Cd4 creERT2 male and DT-treated Foxp3 DTR-GFP/LSL Cd4 creERT2 female mice were likely due to sensing of inflammation assisted by restored Foxp3 expression.

A single cohort of Treg cells provides long-term protection
To test whether the containment of autoimmunity afforded by a single cohort of Treg cells was durable, we monitored tdTomato-labeled and -unlabeled cells in 4-OHTtreated male Foxp3 LSL Cd4 creERT2 R26 Tom and control Foxp3 DTR-GFP Cd4 creERT2 R26 Tom mice for an extended period of time (Fig. 4a). Within a month after treatment, the Foxp3 LSL Cd4 creERT2 R26 Tom mice recovered completely from otherwise lethal disease and continued to gain weight (Extended Data Fig. 7a). At the 4-month time point, only recirculating Treg cells lacking CD73 expression were detected in the thymus, suggesting a lack of continuing thymic Treg output in agreement with our observation that Cd4 creERT2 -mediated recombination ceased within 48-72 hours after 4-OHT administration  34 . The peripheral "4-month-old" Treg pool was well maintained in both lymphoid and non-lymphoid tissues and remained functionally competent ( Fig. 4c; Extended Data Fig. 7c). Nearly all Thy1.1 − GFP + Foxp3 + Treg cells in Foxp3 LSL Cd4 creERT2 R26 Tom mice expressed the tdTomato reporter, whereas Thy1.1 + GFP − "wannabes" and Thy1.1 − GFP − Foxp3 − conventional CD4 T cells in the same mice, as well as Treg and conventional CD4 T cell subsets in control Foxp3 DTR-GFP Cd4 creERT2 R26 Tom mice, contained only a small fraction of tdTomato + fate-mapped cells (Fig. 4d). Thus, Treg cells generated as a single cohort in diseased Foxp3 LSL Cd4 creERT2 mice continued to persist while the other T cell subsets turned over. Impressively, even after 4 months, the reversal of autoimmune disease seemed complete with no tissue pathology observed and T cell activation, effector cytokine production, and myelo-proliferation, at best minimally increased ( Fig. 4e-h; Extended Data Fig. 7d-f). Thus, a single cohort of Treg cells is capable of reversing established inflammation and affording long-term protection against autoimmunity. The rescued mice survived for at least 7 months (Fig. 5a). Even at this time point, the rescued mice remained largely healthy with only moderately increased T cell activation and effector cytokine production and well-maintained Treg populations in both lymphoid and non-lymphoid tissues (Extended Data Fig. 8a-c). Despite mildly elevated serum IgG1and IgE levels in comparison to the 4-week time point, the other Ig isotype levels remained comparable to those in control Foxp3 DTR-GFP/y Cd4 creERT2 mice (Extended Data Fig. 8d). Neither did we observe prominent immune infiltrates in the skin, liver, and small intestine (Extended Data Fig. 8e).
Since TCR specificity and diversity are essential for Treg-mediated control of autoimmunity 35,36 , we assessed the TCR repertoires of long-lived redeemed Treg cells in Foxp3 LSL/y Cd4 creERT2 mice by sequencing their TCRα chains at different time points after Foxp3 restoration. We observed a trend, albeit not statistically significant, toward a slight reduction in TCR diversity, as reflected in the gradually decreasing inverse Simpson indeces (Extended Data Fig. 8f). At later time points (5 and 7 months), the rescued Treg cells contained moderately reduced numbers of total unique clones compared to an earlier time point after the inflammatory disease subsided (1.5 months). The "unevenness" of the TCR repertoire measured by Gini coefficient remained unchanged throughout the entire time course (Extended Data Fig. 8g). These results suggested that the clonal diversity of long-lived redeemed Treg cells was largely preserved even though their TCR richness might have very mildly contracted. Thus, a cohort of restored Treg cells maintained a diverse TCR repertoire, which was likely essential for their ability to confer long-term containment of autoimmune inflammation.

Transcriptional features of long-lived protective Treg cells
The extraordinarily long-term protection against autoimmunity by a single cohort of self-renewing Treg cells suggests that these cells do not exhibit dysfunction typical of chronically stimulated CD4 and CD8 T cells. Thus, we sought to analyze this long-lived Treg population at single-cell resolution using single cell RNA sequencing (scRNA-seq). We compared GFP + Treg cells from Foxp3 LSL/y CD4 creERT2 R26 Tom mice and Foxp3 DTR-GFP/y CD4 creERT2 R26 Tom control mice 7 months after 4-OHT treatment (Fig.   5b). Treg cells in experimental and control mice were efficiently and comparably labeled by tdTomato shortly after 4-OHT administration (Fig. 5c). The percentage of tdTomato + cells among Thy1.1 − GFP + Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice remained high (>80%), demonstrating their impressive persistence. However, among control Foxp3 DTR-GFP Treg cells, tdTomato positivity gradually declined to ~15% at 7 months, most likely due to continuous thymic output of tdTomato − Treg cells (Fig. 5c).
In the scRNA-seq data analysis, we distinguished "old" and "young" control Treg cells from Foxp3 DTR-GFP/y Cd4 creERT2 R26 Tom mice by computationally separating them into tdTomato-positive ("old") and -negative ("young") cells based on the expression of tdTomato transcript by the individual cells. Thy1.1 − GFP + Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice, as well as tdTomato + and tdTomato − control GFP + Treg cells in Foxp3 DTR-GFP/y Cd4 creERT2 R26 Tom mice, exhibited distinct UMAP distribution patterns (Fig. 5d). To better understand the most significant sources of variation among the three Treg cell populations, we performed diffusion map analysis. The first diffusion component (DC1) had a highly similar distribution of gene expression to that of activated Treg gene signature 30 (Fig. 5e). The long-lived Thy1.1 − GFP + Treg cells had a DC1 density resembling that of tdTomato + control Treg cells, both higher than that of tdTomato − cells (Fig. 5f). This result indicates that long-lived Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom and control mice tagged with tdTomato 7 month earlier and their offspring exhibited a similarly activated phenotype. Upon examining genes whose expression patterns highly correlated with DC1, we found transcripts associated with Treg activation (Cd44, Sell, Tnfrsf18, Icos, Ctla4), migration to non-lymphoid tissues (Ccr4, Cxcr3, Ccr6, Itgae), as well as tissue adaptation and suppressor function (Maf, Ahr) (Fig. 5g, Extended Data Fig. 9a) 37,38 . Flow cytometric analysis of CD62L (Sell) and CD103 (Itgae) expression confirmed the scRNA-seq results across multiple tissues, showing sharp reduction in CD62L hi and increase in CD103 + cells among Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice (Extended Data Fig. 9b). Furthermore, the tdTomato + fraction of control Treg cells were enriched for Ly6C − CD103 + and CD44 high CD62L lo cells, consistent with their heightened activation state (Extended Data Fig. 9c). Despite the burden of being the sole Treg population responsible for immunosuppression, the Thy1.1 − GFP + Treg cells did not appear less fit than their "age-matched" counterparts in control mice with continuous Treg turnover. Compared to tdTomato + Treg cells in Foxp3 DTR-GFP/y Cd4 creERT2 R26 Tom mice, Thy1.1 − GFP + Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice exhibited comparable expression of genes associated with T cell exhaustion 39 and apoptosis, and similar levels of Mcl1, an anti-apoptotic gene critical for Treg survival 40 (Fig. 5f). Genes involved in cell cycle progression and various metabolic pathways were also comparably expressed by tdTomato + Treg cells in Foxp3 DTR-GFP/y Cd4 creERT2 R26 Tom mice and Thy1.1 − GFP + Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice (Extended Data Fig. 9d). These shared transcriptional features highlighted the similarities in metabolic status and proliferative potential between the long-lived Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice and "agematched" tdTomato + control Foxp3 DTR-GFP/y Treg cells.
To further compare Thy1.1 − GFP + Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice and long-lived control cells present within the peripheral Treg pool under physiologic conditions, we performed trajectory analysis using the Palantir algorithm, which generates a highresolution pseudotime ordering of cells at different developmental stages 41 . The pseudotime gradient started from resting Treg cells, proceeded towards activated Treg cells, and ended with cells reminiscent of Treg cells residing in non-lymphoid organs in agreement with a previous report 42 (Fig. 5h). The analysis successfully recapitulated known gene expression dynamics during Treg activation including downregulation of Tcf7, Sell, and Klf2, and upregulation of Foxp3, Ctla4, and Ikzf2 (Fig. 5i). Next, we compared the pseudotemporal ordering of Thy1.1 − GFP + Treg cells from Foxp3 LSL/y Cd4 creERT2 R26 Tom mice derived from cells whose Foxp3 expression was "restored" 7 months ago, with that of similarly "old" tdTomato + and of "young" tdTomato − Treg cells from control Foxp3 DTR-GFP/y Cd4 creERT2 R26 Tom mice. Both "old" Treg populations were positioned late in the inferred differentiation trajectory in comparison to tdTomato − Treg cells-consistent with their activated phenotype-and were characterized by comparablely high pseudotime values (Fig. 5j). Thus, Thy1.1 − GFP + Treg cells in Foxp3 LSL/y Cd4 creERT2 R26 Tom mice closely resembled a subset of long-lived cells present in the normal Treg cell pool under physiologic conditions.
To gain further insights into the long-term persistence of Thy1.1 − GFP + Treg cells in Foxp3 LSL Cd4 creERT2 R26 Tom mice, we performed a refined clustering analysis of the scRNA-seq dataset. Among the 13 clusters identified, clusters 0 and 3 were primarily composed of tdTomato − Foxp3 DTR-GFP control Treg cells, whereas clusters 1, 6, 7 and 8 were enriched for Thy1.1 − GFP + Treg cells (Fig. 6a). Importantly, cells from cluster 1 had higher expression of IL-2-Stat5 and Wnt-β-catenin pathway gene signatures (Fig. 6b), which have been implicated in Treg cell maintenance and selfrenewal 43,44,45 . Moreover, these cells were lower in pseudotime values compared to most Thy1.1 − GFP + Treg cells, suggesting that they were less differentiated and had the potential to give rise to the rest of the cells and sustain the Treg pool (Fig. 6c). Gene expression analysis revealed transcripts enriched (Ifngr1, Epcam, Lrrc32, Il4ra, etc.) and depleted (Sell, etc.) in cluster 1 cells (Fig. 6d, Supplementary Table 1). Using flow cytometry, we corroborated these results by demonstrating a marked enrichment of an IL-4Rα hi IFNγR1 hi Epcam + GARP hi CD25 hi (γREG + ) cell subset within activated CD62L lo rescued Treg population in Foxp3 LSL Cd4 creERT2 R26 Tom mice ( Fig. 6e-f, Extended Data Fig. 10a-e). In normal mice, these cells were also present, albeit at lower frequencies, within Treg population in secondary lymphoid organs and at even lower frequencies in non-lymphoid tissues, such as the liver and lung, but were virtually undetectable among newly differentiated CD73 − thymic Treg cells (Fig. 6g). γREG + Treg cells were enriched in the parenchyma of highly vascularized organs such as spleen and lung, as evidenced by their higher frequencies among cells not labeled by intravenously administered CD45 antibody. This suggested that these cells were largely non-circulatory and likely contributed to local maintenance of the Treg pools (Extended Data Fig. 10f). To compare the capacity of perinatal and adult Treg populations to give rise to γREG + Treg cells, we labeled Treg cells in healthy 2-week-old and 8-week-old Foxp3 DTR-GFP Cd4 creERT2 R26 Tom mice by 4-OHT treatment and assessed the frequencies of γREG + Treg cells within tdTomato + and tdTomato − Treg subsets 4 months later. In mice treated as perinates, tdTomato + Treg cells harbored higher frequencies of γREG + Treg cells than their tdTomato − counterparts (Fig. 6h). In contrast, tdTomato + Treg cells time-stamped during adulthood were not enriched for γREG + Treg cells over their tdTomato − counterpart, and similar percentages of γREG + cells were found among tdTomato − Treg cells in perinatally labeled mice (Fig. 6h). These observations were independently confirmed by similar time-stamping of Treg cells in young vs. adult Foxp3 creERT2 R26 Tom mice (Fig. 6h). These results were consistent with the enrichment of γREG + Treg cells among Treg populations in secondary lymphoid organs and non-lymphoid tissues in unmanipulated 2-week-old vs. 8-week-old mice (Extended Data Fig. 10g). Our results suggest γREG + Treg cells, capable of persisting in peripheral lymphoid and non-lymphoid organs, are selectively overrepresented within the Treg population generated early in life.

Discussion
The adaptive immune system is characterized by unlimited antigen recognition specificity, amplification of innate immune responses, novel effector functions, and memory formation, affording vertebrates with a versatile anticipatory defense against rapidly evolving infectious agents. This superior protection is confounded by a major fitness constraint due to the threat of autoimmune inflammation posed by the inherent self-reactivity of T cells. The importance of Treg cells in forestalling autoimmunity has been most vividly demonstrated by wide-ranging clinical manifestations of human monogenic disorders resulting from Treg deficiency or dysfunction due to FOXP3, STAT5B, IL2RA, and LRBA mutations or CTLA4 haplo-insufficiency 46,47 . In IPEX patients, these manifestations include endocrinopathies (diabetes, thyroiditis, pancreatitis, adrenal dysfunction), hepatitis, enteropathies (autoimmune gastritis, IBD, celiac disease), skin disorders (exudative dermatitis, alopecia), food allergy, hyper-IgE syndrome, autoimmune hematologic disorders (autoimmune thrombocytopenia, hemolytic anemia), myelo-and lymphoproliferation (splenomegaly, lymphadenopathy), polyneuropathy, as well as pulmonary and nephrotic syndromes 48 . In mice, Treg deficiency or depletion causes equally widespread and fatal autoimmune inflammatory disease 13 .
However, the potent immunosuppressive function of Treg cells indispensable for preventing autoimmune inflammation can in turn compromise protective immunity against infections. This provided rationale for the concept that Treg cells are ineffectual in the inflammatory settings of infections. Indeed, numerous studies showed that infection-associated inflammatory milieu can cause downregulation of Foxp3 expression by Treg cells, impede their suppressor and proliferative capacity, or even confer pro-inflammatory properties, i.e. effector cytokine production; furthermore, pro-inflammatory cytokines, IL-6 and IL-1 in particular, can make effector cells refractory to Treg-mediated suppression 14,16,18,19,49,50 . On the other hand, some studies suggested that Treg cells are capable of modulating virusspecific immune responses and limiting associated pathologies 51,52,53,54,55,56 . In all these studies, however, Treg cells were present from the onset of infection, making it impossible to discriminate their activity prior to, or after the onset of infection-induced inflammation.

Thus, the ability of Treg cells to function in infection-induced inflammation remains an open question.
Since inflammation elicited by various infectious, metabolic, and genetic causes, including Treg deficiency, shares principal cellular and soluble mediators, a corollary to the above concept is the notion that in established autoimmune inflammation, Treg activity is also expected to be severely attenuated or even lacking. In this regard, autoimmune and inflammatory pathologies were reported to exert varying effects on Treg numbers. In contrast to the aforementioned monogenic Treg deficiency-linked disorders, many studies of polygenic autoimmune diseases have found unchanged or even increased frequencies of Treg cells at inflammatory sites and in circulation 57 . Meanwhile, analyses of Treg cells isolated from autoimmune patients suggested that their function is impaired in the inflammatory environment 21,22,23,57,58,59 . However, considering numerous genetic polymorphisms linked to the pathophysiological manifestations of polygenic autoimmune diseases, the reported inferior Treg functionality can be one of these sequelae. Indeed, an enrichment for such autoimmunity-associated single nucleotide polymorphisms has been observed within Treg-specific cis-regulatory elements 10, 60, 61 . As a potential counterargument to the above findings, adoptive transfer of Treg cells after the initiation of T cell-induced colitis was shown to cause its amelioration over time in an IL-10-dependent manner, highlighting their potential to control local inflammation elicited upon homeostatic expansion of a relatively small number of naïve CD4 T cells in lymphopenic mice 62, 63 . Thus, it remained possible that the reparative activity of Treg cells observed in this setting may be specific to the colon aided by its continuous epithelial cell turnover, or particular to the adoptive transfer model. By genetically introducing a limited Treg pool in Foxp3 LSL mice, we demonstrated that Treg cells are able to function in settings of systemic autoimmune inflammation, reverse the fatal disease and tissue pathology, and offer long-term protection. The reversal of inflammation was not a simple diversion of a large number of pro-inflammatory effector T cells into Treg cells because indistinguishable fatal autoimmunity observed in Foxp3-deficient mice harboring Treg "wannabes" and Foxp3 DTR neonates subjected to chronic ablation of Treg cells suggests that Treg "wannabes" do not provide notable non-redundant contribution to the disease progression 5,8,13 . It is noteworthy that upon generation of functional Treg cells in Foxp3 LSL mice, the reduction in myeloid cells was observed with a markedly slower kinetics than the decrease in activated T cell numbers and responses, suggesting that the reversal of myelo-proliferation was largely an indirect effect of suppression of T cell activation. Nevertheless, a parallel direct suppression of myeloid cell activation and survival by Treg cells may also contribute to the recovery of Foxp3 LSL mice from autoimmunity. The ability of Treg cells to suppress established inflammation was not limited to young mice but was also observed in adult animals. Notably, Treg cells introduced into the inflammatory environment suppressed Th2 autoimmunity and associated increased serum IgG1 and IgE levels, as efficiently as Th1 and Th17 responses, even though Th2 responses are known to be the most sensitive to diminished Treg numbers or functionality 26,64 .
The observed normalization of lympho-and myeloproliferation, acute phase response, and reversal of tissue inflammation highlighted the ability of Treg cells to function in a cytokine storm. Rather than impeding functionality, we found that exposure to this highly inflammatory milieu endowed the newly generated Treg cells with heightened suppressor activity in comparison to Treg cells from healthy control mice, while the latter and Treg cells generated upon a similar conversion of Treg "wannabes" in healthy heterozygous female mice had indistinguishable suppressor activity. Treg cells employ multiple and partially redundant modes of suppression, including secreted immunomodulatory factors, IL-2 consumption, and ATP-to-adenosine conversion, and may directly partake in tissue regeneration and repair via the production of amphiregulin 65,66 . Accordingly, redeemed Treg cells in Foxp3 LSL mice exhibited increased expression of IL-2R, IL-10, galectin-1 and -3, Fgl2, CTLA4, CD39, ST2, and amphiregulin. While we cannot unambiguously pinpoint the inflammation-sensing pathways responsible for the potentiation of their function, prominent STAT5 activation gene signature in these cells implicates common γ-chain receptor signaling cytokines including IL-2. Accordingly, administration of therapeutic IL-2 formulations and expression of an active form of STAT5b in Treg cells promote their expansion and superior suppressor function 31,67,68 . Notably, the observed suppressor activity of Treg cells in inflammatory settings was not limited to recent thymic emigrants as efficient suppression of autoimmunity in systemically inflamed mice was observed upon pharmacological thymic export blockade. The reversal of inflammatory disease and longterm maintenance of health by a single pool of Treg cells was enabled by its stability, which may be aided by a subset of preferentially tissue-residing cells with increased expression of IFNγR, IL-4Rα, EpCAM, CD25, and GARP. Based on gene set enrichment and pseudotime analyses, these cells may contribute to long-term Treg maintenance by giving rise to terminally differentiated progenies locally. Interestingly, time-stamped "old" peripheral Treg population in lymph nodes of adult mice was enriched for γREG + Treg cells in comparison to other sites such as the spleen or liver, raising the possibility of specific niches that favor their generation or maintenance.
Collectively, we show that Treg cells can function in established severe inflammation and reverse all known types of inflammation, and that a single cohort of Treg cells can afford long-lasting protection against autoimmunity. Our findings provide general rationale and support for the emerging efforts to develop adoptive Treg therapy not only for intrauterine and neonatal IPEX syndrome and other monogenic Treg deficiencies, but also for a broad spectrum of autoimmune and inflammatory disorders.

Mice
Experiments in this study were approved by the Sloan Kettering Institute (SKI) Institutional Animal Care and Use Committee under protocol 08-10-023 and conducted in compliance with institutional ethics guidelines. Mice were housed at the SKI animal facility under SPF conditions on a 12-hour light/dark cycle with free access to water and regular chow diet. The average ambient temperature is 21.5°C and the average humidity is 48%. All control and experimental animals were age-matched, and littermates were used as controls unless otherwise indicated. Age, sex, and numbers of animals used in each experiment are indicated in the respective Figure Legends.

Generation of Foxp3 LSL mice
The targeting construct was made by first subcloning an 8.5 kb SphI fragment harboring the Foxp3 genomic sequence from exon -1a to exon 7 obtained from the RP23-143D8 cosmid into a cloning vector carrying a Pgk-DTA-polyA cassette allowing for negative selection of random genomic integration. The FRT-PGK-neo-polyA-FRT positive selection cassette was then cloned into the DraIII site within the intron between exons -1b and 1, and the loxP-Thy1.1 coding sequence-triple-tandem SV40 polyA-eGFP coding sequence cassette was cloned into the AvrII site within exon 1. The Thy1.1 and eGFP sequences are preceded by a start codon (ATG). The targeting construct was then electroporated into albino C57BL/6 ES cells. After neomycin selection, Southern blotting and karyotyping, correctly targeted clones were injected into WT C57BL/6 blastocysts. The resulting chimeric mice were bred to albino C57BL/6 mice. Founders identified based on the coat color and genotyping were mated to a Flp deleter strain of mice to remove the neo cassette.
Diphtheria toxin (DT, List Biological Laboratories, 150) was dissolved in PBS. 1μg (first dose) or 250 ng (subsequent doses) was injected intraperitoneally for each mouse. For tamoxifen administration, 20 mg of tamoxifen (Sigma-Aldrich, T5648) was resuspended in 1 mL corn oil (Sigma-Aldrich, C8267) by rotating and tilting at 37°C until fully dissolved. Each mouse was orally gavaged with 4 mg of tamoxifen per treatment.
4-OHT was used whenever possible because it bypasses the conversion of tamoxifen to 4-hydroxytamoxifen in the liver. Compared to tamoxifen, 4-OHT offers a much sharper pharmacokinetics and enables a highly synchronized labeling of cells. In addition, 4-OHT has a much shorter half-life than tamoxifen, particularly in adults. Therefore, tamoxifen instead of 4-OHT was used for in vivo suppression assays (Extended Data Fig. 1g) and in studies of adult female mice (Fig. 2, Fig. 3 h-j, and Extended Data Fig. 5) to achieve sufficient recombination of the Foxp3 LSL allele. FTY720 (Sigma-Aldrich, SML0700-5MG) stock solution was made by reconstituting in dimethyl sulfoxide (Sigma-Aldrich, D8418-250ML) at a concentration of 20 mg/ml. 0.8 μg/g body weight of FTY720 diluted to 0.1 mg/ml in 2% β-hydroxypropanylcyclodextrin (Sigma-Aldrich, H5784-10ML) was administered intraperitoneally.

Reagents and antibodies
The following antibodies and streptavidin were used in this study for flow cytometry, with clones, venders, catalog numbers, and dilutions indicated in the parentheses: anti-Siglec-F

Measurement of serum albumin
Serum albumin level was measured by Laboratory of Comparative Pathology, SKI, using the Albumin kit (Beckman Coulter, OSR6102), according to manufacturer's instruction.

In vitro suppression assay
A 2-fold titration series of FACS-sorted Treg cells starting from 40,000 cells/well was set up in U-bottom 96-well plates. 40,000 FACS-sorted, CellTrace Violet (ThermoFisher, C34571)labeled naïve CD4 T cells and 100,000 erythrocyte-lysed splenocytes from Tcrb −/− Tcrd −/− mice as antigen-presenting cells were then added to each well. α-mouse CD3 (145-2C11, BioXCell, BE0001-1) was then added to a final concentration of 1 μg/mL. Cells were incubated in a final volume of 200 μL complete RPMI w/ 10% FBS and with 5% CO 2 at 37°C for 72 hours before analysis. Cells that have had more than 4 rounds of CTV dilution were considered divided for calculating Treg cell-mediated suppression using the following formula:  75 was used to perform differential gene expression analysis across different conditions. A cutoff of 0.05 was set on the obtained FDR-adjusted p-values to get the significant genes of each comparison. All detectable genes were rlog-normalized and then used for the principal component analysis.

Single cell RNA sequencing and data analysis
Library Preparation and sequencing-Library preparation and sequencing for the scRNA-Seq of doubly-FACS-sorted Treg cells isolated from the secondary lymphoid organs of 7 months post-4-OHT treatment Foxp3 DTR-GFP (cells pooled from 5 mice) and Foxp3 LSL Cd4 creERT2/+ mice (cells pooled from 4 mice) were performed by the Single Cell Data Pre-processing-Fastq files were processed using Cell Ranger v3.0 (10x Genomics) and reads were aligned to the mouse genome mm10 from ENSEMBL GRCm38 that was modified to include sequences corresponding to the coding region and the 3'UTR of the R26 Tom allele. Cells containing over 5% mitochondria-derived transcripts were filtered out, resulting in 3,634 Foxp3 DTR-GFP cells and 3,390 Foxp3 LSL cells that passed QC metrics, with a median of 3,580 molecules/cell. Cells with total molecule counts below 1000, as determined by the lower mode of the distribution of total molecules per cell, were additionally filtered out to remove putative empty droplets. Genes that were expressed in more than 10 cells were retained for further analysis. The resulting count matrices from both samples were then combined, resulting in a final set of 7,024 cells x 12,432 genes, and normalized to median library size, where library size is defined as total molecule counts per cell. The normalized data are then log transformed as log (counts + 1) for downstream analysis.
Principal component analysis-For dimensionality reduction and visualization of data, we further excluded genes with very low or very high expression of transcripts (log average expression <0.02 or >3 and dispersion >0.15), and principal component analysis was then performed on the log-transformed normalized data. Using 40 principal components, where the number of principal components was determined by the knee-point in eigenvalues, yielded a good representation of the variability in the data.
MAGIC imputation-To account for missing values in scRNA-seq due to a high dropout rate, we employed MAGIC v0.1.1, a method of "de-noising" and imputing missing expression values through data diffusion between cells with similar covariate gene relationships 76 . We constructed the diffusion map matrix using k = 30, ka = 10, and t = 6 as input parameters, where t specifies the number of times the affinity matrix is powered for diffusion.
Diffusion components and pseudotime calculation-Instead of constructing a tSNE map using 40 PCs, we followed the strategy outlined by Setty et al. 41 In order to characterize potential pseudotime non-linear trajectories and to visualize single-cell gene expression in a UMAP embedding of diffusion components. Based on the eigen gap, we chose to use 15 diffusion components for downstream analysis in Palantir v1.0.0 and for calculating diffusion distances. We scaled each included diffusion component by the factor λ/(1-λ) where λ is the associated eigenvalue, to reflect 'multi-scale' diffusion distances.
Then, we calculate each cell position in pseudotime based on modeling cell fate in a continuous probabilistic model.
Clustering and gene ranking-Clustering of cells was performed using PhenoGraph v1.5.7 77 setting k = 15 nearest neighbors. A cluster was removed because of its disparity with the rest of the data (t-SNE projected this cluster as a separate component that comprised cells from both populations), and those cells also had a relatively lower number of total molecules compared with other populations. Significant differentially expressed genes in each cluster were identified using t-test (where the variance of small groups is overestimated), which was implemented in Single-Cell Analysis in Python (Scanpy) v1.7.2 78 with default parameters.
tdTomato expression and gene set module score calculation-Because of a high dropout rate of single cell sequencing, we performed MAGIC imputation of tdTomato expression (as described above) only for Foxp3 DTR-GFP cells since the overwhelming majority of the Foxp3 LSL cells were tdTomato + which could potentially cause overimputation. A cutoff of 1.04 was set for the imputed tdTomato expression where any cells with higher expression were categorized as tdTomato + (~15% of cells) and those with lower expression as tdTomato − (~85% of cells) in agreement with flow cytometric measurements. Gene set module scores for Il2-Stat5 (GSEA HALLMARK_IL2_STAT5_SIGNALING) and Wnt/β-catenin (GSEA HALLMARK_WNT_BETA_CATENIN_SIGNALING) were calculated with the AddModuleScore function in Seurat v 3.1.5 79 using the default parameters.

TCR sequencing and data analysis
Bulk sequencing of the TCRα chain was performed using a 5' RACE-based method as previously described 36 . Briefly, 70,000 rescued Treg cells (TCRβ + CD4 + GFP + Thy1.1 − ) from Foxp3 LSL/y Cd4 creERT2 mice were FACS-sorted from pooled spleen and lymph nodes at different time points post 4-OHT administration. Total RNA was extracted using the RNAeasy Plus Micro Kit (Qiagen 74034) according to the manufacturer's instructions. cDNA was synthesized using the SMARTScribe Reverse Transcriptase (Clontech 639537) with a mixture of oligo(dT) 24 and primers targeting the mouse TCRα constant regions. A template switching DNA-RNA hybrid oligo containing 12 random nucleotides was used to hybridize onto the 3' end of the first stand cDNA and to barcode the individual cDNA molecules. After removal of the hybrid oligo with Uracil-DNA glycosylase (New England Biolabs M0280), the cDNA was further PCR amplified to introduce sample barcodes, sequencing primer binding sites, as well as the Illumina P5 and P7 sequencing adaptors. The final PCR products were separated on a 1% agarose gel and a single band around 700 bp was cut and purified using the NucleoSpin Gel and PCR Clean-up Kit (Clontech 740609). After PicoGreen quantification and quality control by Agilent TapeStation, libraries were pooled in equimolar ratios and run on a MiSeq in a PE200 run, using the MiSeq Reagent Kit v3 (600 Cycles) (Illumina). The loading concentration was 7-18 pM and a 10-20% spike-in of PhiX was added to the run to increase diversity and for quality control purposes (libraries were sequenced 3 times; loading concentration and PhiX spike-in amounts were adjusted based on initial performance). The runs yielded an average of 815K reads per sample.
The fastq files containing the TCR sequencing reads were aligned using the MiXCR software v3.0.13 to reference the V, D and J genes of mouse T cell receptors 80 . MiXCR was also used to assemble clonotypes using alignments obtained from the previous step in order to extract the highly variable CDR3 region. The resulting clonotypes were pre-processed using VDJtools 1.2.1 81 in two steps. The first is a frequency-based correction to eliminate erroneous clonotypes. The algorithm searched the sample for clonotype pairs that differ by up to 2 mismatches. In case the ratio of the smallest to the largest clonotype sizes was lower than a specified threshold of 0.05^(number of mismatches), correction was performed by discarding the smaller clonotypes. The second step was to filter non-functional clonotypes. Specifically, the ones that contained a stop codon or frameshift in their receptor sequences were discarded. The resulting clonotypes and clone sizes were used to calculate the Simpson index and Gini coefficient. Two different clonotype matching strategies were used: 1) V, J and CDR3 nucleotide sequences and 2) the CDR3 amino-acid sequence.

Statistics
Statistical significance was determined using tests indicated in the respective figure legends. P-values for t-test and Mantel-Cox test on flow cytometric and survival data were calculated with GraphPad Prism 7 and had been corrected for multiple hypothesis testing using the Holm-Sidak method, when applicable. P-values for ANOVA were computed with R for Fig.  2b and GraphPad Prism 7 elsewhere and had been corrected for multiple hypothesis testing using the Tukey method (when using R and Prism), the Dunnett's method or the Sidak method (when using Prism, according to its recommendations). P-values for Kolmogorov-Smirnov test were calculated with R. Throughout the entire study, error bars represent mean ± s.e.m., and the following notation was used to report statistical significance: ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Data availability
All sequencing data generated in this study can be accessed at GEO under accession number GSE179710. The custom mouse genome used for scRNA-seq analysis, which was generated by adding the tdTomato sequence to GRCm38 (https://www.ncbi.nlm.nih.gov/ assembly/GCF_000001635.20/), is also available at GEO.

Code availability
All custom scripts are available upon request to the corresponding authors.  Mice were treated with 4-OHT on postnatal day 14 and analyzed 7 months later. a-c, Frequencies of Treg cells (a) and proliferating, activated, and cytokine-producing conventional CD4 (b) and CD8 (c) T cells. Two-tailed unpaired t-tests with multiple hypothesis testing correction using the Holm-Sidak method. pLN, peripheral (brachial, axillary, and inguinal) lymph nodes; mLN, mesenteric lymph nodes; LP, lamina propria. d, Serum antibody levels. Scales were kept the same as in Figure 1g. Two-tailed unpaired t-test.

Extended Data
e, Representative images of haematoxylin and eosin-stained sections of the indicated organs. Images are representative of 5 Foxp3 DTR-GFP/y and 5 Foxp3 LSL/y mice. f, Clonal diversity of the TCRα repertoire of the long-lived "redeemed" Treg cells from Foxp3 LSL/y Cd4 creERT2 mice at indicated time points after restoring Foxp3 expression upon 4-OHT treatment. The inverse Simpson Index was calculated based on the clone size distribution using clonotypes defined by full nucleotide sequence (left) or CDR3 amino acid sequence (right). g, Total number of unique clones (left) and Gini coefficient (right) of the TCRα repertoire of the long-lived "redeemed" Treg cells. Clonotypes were defined by using the full nucleotide sequence. One-way ANOVA with Dunnett's multiple hypothesis test. All error bars denote mean ± s.e.m. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.